How does the General Theory of Cardiac Chronotropy and Inotropy Apply to Aging? Both our earlier and very recent studies have contributed seminal perspectives toward understanding age-associated deterioration of both cardiac contractility and heart rate in both humans, and in animal models. Our studies of healthy participants in the Baltimore Longitudinal Study of Aging (BLSA) demonstrated substantial age-associated changes in the ability to increase heart rate and reduce end systolic volume in response to graded exercise stress. (Of note, only the latter, but not the former, can be improved by physical conditioning.) We subsequently demonstrated that deficits both in contractility and heart rate in humans are due, in part, to reduced -AR stimulation response of cAMP-PKA. 1. Contractility In isolated rat cardiac ventricular muscle, we had directly demonstrated a reduction in the Ca2+ cycling and contractile response to -AR stimulation. The relaxation time of the Ca2+ transient and contraction in the absence of -AR stimulation were prolonged, that we traced to a reduced expression of SERCA2. The AP was also markedly prolonged, due to age-associated changes in L-type Ca2+ and K+ currents. In single VM, in addition to numerous properties of surface membrane ion channels, we documented an age-associated reduction in the VM Ca2+ clock, manifest as a prolonged time for restitution of the excitation- Ca2+ release-contraction coupling process, was due to a prolonged restitution time for SR Ca2+ release via RyRs in response to activation by an L-type Ca2+ current. We also showed that SR Ca2+ loading and the amplitude of the Ca2+ transient were preserved in myocytes from the old heart, by virtue of the prolonged AP. 2. Heart rate Our discovery of heart rate regulation by a coupled-clock system in rabbit SANC enabled progress, finally (after 25 years), on the elucidation of potential cellular mechanisms of the age-associated reduction in chronotropic reserve. Specifically, our conceptual breakthrough that regulation of basal pacemaker cell automaticity requires Ca-PKA-CaMKII signaling regulated by PDE and phosphatase activities, and that stimulation of receptors merely extended this regulation, led us to hypothesize that mechanisms involved in the deterioration of mechanisms that regulate of intrinsic heart rate that accompanies advancing age and that those regulating heart rate reserve may involve a slowing of the Ca2+ clock of SANC and linked, in some respects, at least, to the age-associated deterioration of mechanisms that regulate Ca2+ cycling and contractility in VM. In sinoatrial tissue under basal state the beating rate is lower in aged mice, but the rhythm variability is higher. The maximal responses of sinoatrial tissue beating rate and rhythm variability to autonomic receptor stimulation remain intact in mice but both are less sensitive in advanced age. Changes in beating rate in response to autonomic receptor stimulation occur concurrently with changes in rhythm variability; i.e., increase in beating rate is associated with decrease in rhythm variability and vice versa. Recently, we have learned how to successfully isolate single sinoatrial node cells (SANC) from young, old C57B1 and transgenic mice. We tested our hypothesis that changes in spontaneous sarcoplasmic reticulum (SR) Ca2+ cycling and SANC ability to response to the cAMP-PKA-dependent signaling might play a significant role in the age-related SAN dysfunction and reduced beating rate with age. Our initial results indicate that: the spontaneous AP firing rate of single SANC declines with age; the maximum Ca2+ release flux in response to an AP declines with age; and the kinetics of relaxation of the Ca2+ transient become prolonged with age. We measured spontaneous local Ca2+ releases (LCRa) in single permeabilized SANC from young and old C57B1 mice, in conditions of tight control of intracellular free Ca2+ levels. We discovered a reduced SR Ca2+ load, smaller size, duration and number of LCR events in old vs. young SANC in basal conditions at constant free Ca2+. Moreover, when we activated of cAMP-PKA-Ca2+ pathway by phosphodiesterase inhibitor (IBMX) we found an increase in average LCR characteristics (size, duration, amplitude) and percent cells with periodic LCRs in young vs. old. PLB phosphorylation at Ser16 (PLB/total PLB) immunolabeling in control did not differ with age, but increased 2.20.2 fold after incubation with 10 M IBMX in young but did not increase in old SANC. We conclude that intrinsic SR Ca2+cycling and its response to PDE inhibition decline with age, and that this may explain why the aged heart cannot beat as fast as the young heart. Our results suggest that the intrinsic cAMP-PKA-Ca2+ signaling is deficient in the aged mice. This deficiency may result from: 1) reduced amount or function of Ca2+ cycling proteins e.g. SR Ca2+ pump, PLB, RyRs. and/or 2) reduced phosphorylation of Ca2+ cycling proteins e.g. PLB, in response to an increase in cAMP-mediated-PKA dependent phosphorylation. Future plans are: 1) to employ western blotting in cells from young and old SANs to measure Ca2+ cycling protein density; 2) to directly measure the downstream signaling of cAMP-PKA- Ca2+ pathway i.e. PKA-dependent protein phosphorylation and phosphatase activity.